Chimeric papillomavirus-like particles

ABSTRACT

The present invention provides a papillomavirus-like particle, characterized as having conformational epitopes, comprising a papillomavirus L1 product and a papillomavirus L2 fusion product; and related synthetic DNA molecules, host cells, methods and vaccines.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims the benefit of priority ofU.S. patent application Ser. No. 11/807,004, filed May 25, 2007, whichis a continuation and claims the benefit of priority of patentapplication Ser. No. 11/022,091, filed Dec. 22, 2004, now abandoned,which is a continuation and claims the benefit of priority of U.S.patent application Ser. No. 10/741,613 filed Dec. 19, 2003, nowabandoned, which is a continuation and claims the benefit of priority ofU.S. patent application Ser. No. 10/405,264, filed Apr. 1, 2003, nowabandoned, which is a continuation and claims the benefit of priority ofU.S. patent application Ser. No. 09/878,840, filed Jun. 11, 2001, nowabandoned, which is a continuation and claims the benefit of priority ofU.S. patent application Ser. No. 09/170,129, filed Oct. 12, 1998, nowabandoned, which is a continuation and claims the benefit of priority ofU.S. patent application Ser. No. 08/781,084, filed Jan. 9, 1997, nowU.S. Pat. No. 5,855,891, which is a divisional and claims the benefit ofpriority of U.S. patent application Ser. No. 08/319,467, filed Oct. 6,1994, now U.S. Pat. No. 5,618,536, which is a continuation-in-part andclaims the benefit of priority of U.S. patent application Ser. No.08/032,869, filed Mar. 16, 1993, now U.S. Pat. No. 5,437,951, which is acontinuation-in-part and claims the benefit of priority of U.S. patentapplication Ser. No. 07/941,371, filed Sep. 3, 1992. These applicationsand all patent applications, patents and publications cited hereunderare incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to chimeric papillomavirus-like particlesand related synthetic DNA molecules, host cells, methods and vaccines.

BACKGROUND OF THE INVENTION

Papillomaviruses infect the epithelia of humans and a wide variety ofanimals, where they generally induce benign proliferation at the site ofinfection. However, in some cases the lesions induced by certainpapillomaviruses undergo malignant progression. There is a strongassociation between malignant progression of human genital lesions andcertain human papillomavirus (HPV) types, such as HPV16. Infection byone of these types is considered the most significant risk factor in thedevelopment of cervical cancer, one of the most common cancers of womenworldwide (zur Hausen, H. 1991 Science 254:1167; Schiffman, M. H. 1992J. Natl. Cancer Inst. 84:394). The majority of cervical carcinomascontain and express HPV early genes, such as E6 and E7, and these geneshave been shown to have potent transforming and immortalizing activityin cultured cells (Werness, B. A., Munger, K. & Howley, P. M. 1991Advances in Oncology, eds. DeVita V. T., Hellman, S. & Rosenberg, S. A.,Lipponcott, Philadelphia, pp. 3-18).

Papillomaviruses are non-enveloped double-stranded DNA viruses about 55nm in diameter with an approximately 8 kb genome in the nucleohistonecore (Baker, et al. 1991 Biophys J 60:1445). The capsids are composed oftwo virally-encoded proteins, L1 and L2, that migrate on SDS-PAGE gelsat approximately 55 kDa and 75 kDa, respectively (Mose Larson et al.1987 J. Virol. 61:3596). L1, which is the major capsid protein, isarranged in 72 pentameters which associate with T=7 icosahedralsymmetry. The function and position within the virion of L2 are unclear(Baker, et al. 1991 Biophys J 60:1445).

The L1 protein has the capacity to self-assemble so that large amountsof virus-like particles (VLPs) may be generated by expression of the L1protein from a number of species of papillomavirus in a variety ofrecombinant expression systems (Hagensee et al. 1993 J. Virol. 67:315;Kirnbauer et al. 1992 PNAS USA 89:12180; Kirnbauer et al. 1993 J. Virol.67:6929; Rose et al. 1993 J. Virol. 67:1936). Although not required forassembly, L2 is incorporated into VLPs when co-expressed with L1 (L1/L2VLPs) in cells.

Immunization of rabbits with native virions or L1 VLPs, but not withnon-assembled L1 expressed in E. coli, induces high titers ofneutralizing serum antibodies (Christensen, N. D. and Kreider, J. W.1990 J. Virol. 64:3151; Kirnbauer et al. 1992 PNAS USA 89:12180;Pilacinski et al. 1984 Bio/Technology 2:356; Segre et al. 1955 Am. J.Vet. Res. 16:517). The polyclonal and monoclonal antibodies generatedagainst native particles recognize conformationally dependent epitopes(Christensen, N. D. and Kreider, J. W. 1993 Virus Res. 28:195;Christensen et al. 1990 J. Virol. 64:5678; Christensen et al. 1991Virology 181:572).

Neutralizing antibodies generated against VLPs also recognizeconformationally dependent epitopes. Using infectious BPV1, which can bereadily obtained from bovine lesions, and a quantitative in vitro BPV1infectivity assay (Dvoretzky et al. 1980 Virology 103:369), workersshowed VLPs from bovine papillomavirus induced high levels ofneutralizing antibodies (Kirnbauer et al. 1992 PNAS USA 89:12180). Theneutralizing antibodies were directed against conformationally dependentepitopes, in that denaturation of the particles prior to immunizationabolished the ability of the preparation to induce neutralizing activity(id.).

When the L1 gene of a HPV16 isolate derived from a nonprogressed lesionwas used to express the L1 major capsid protein in insect cells viarecombinant baculoviruses, L1 self-assembled into VLPs at a yield 3orders of magnitude higher than what had been obtained using L1 derivedfrom the prototype HPV16 (originally isolated from a cancerous lesion),and formed VLPs that were morphologically similar to native virions(Kirnbauer et al. 1993 J. Virol. 67:6929). DNA sequence comparisonidentified a single nonconserved amino acid change to be responsible forthe inefficient self-assembly of the prototype L1 (id.). The L1 of theassembly-competent clone is thus considered to be the wild-type gene,and the prototype L1 of the assembly-defective clone a mutant.

Using HPV16 VLPs of the wild-type L1 protein as antigens, an ELISA wasdeveloped that detected serum antibodies in patients infected with HPV16(Kirnbauer et al. 1994 J. Natl. Cancer Inst. 86:494). In contrast,neither denatured HPV16 particles nor preparations of the prototype L1protein could detect these antibodies (id.). These results demonstratethat the prototype L1 protein does not present conformational epitopes.

Rabbit serum raised against self-assembled wild-type HPV16 L1/L2virus-like particles was discovered to prevent HPV16 VLP binding to cellsurface molecules (Roden et al. J. Virol., in press, (November, 1994)).In contrast, serum raised against the prototype strain of HPV16 L1/L2did not prevent such binding (id.). The data show that the prototypeHPV16 strain lacks conformational epitopes.

Rabbits immunized with intact cottontail rabbit papillomavirus (CRPV)virus-like particles composed of L1 or L1/L2 were protected fromsubsequent experimental challenge by infectious CRPV (Breitburd et al.,12th International Papillomavirus Workshop in Amsterdam, in press,(October 1994)). In contrast, those immunized with denatured particleswere not protected (id.). These findings are consistent with theconclusion that VLPs presenting conformational epitopes are able toinduce protective immunity.

VLPs composed of capsid proteins are attractive candidates forprophylactic vaccines to prevent papillomavirus infection. However, itis unlikely that these VLP vaccines would have therapeutic effectsagainst established papillomavirus infections. The capsid proteins,unlike E6 and E7, are not detectably expressed in progressed lesions orin infected basal epithelial cells, which are the presumed targets inimmune regression of papillomas.

There is evidence from experimental models that immunity againstpapillomavirus proteins other than L1 and L2 might help controlpapillomavirus infection. Since E6 and E7 are selectively maintainedduring oncogenic progression, there is the possibility that peptidesderived from these oncoproteins could serve as targets for cell-mediatedimmune responses to HPV-containing tumor cells. Studies in animal modelssuggest the E7 protein of HPV16 acts as a tumor rejection antigen (Chenet al. 1991 PNAS USA 88:110; Feltkemp et al. 1993 Eur. J. Immunol.23:2242). Moreover, the frequency of HPV infection, persistence of HPVinfection, and risk of developing cervical cancer and other HPV-relatedcancers is increased in patients with depressed cellular immunity(Allout et al. 1989 Br. Med. J. 298:153; Laga et al. 1992 Int. J. Cancer50:45). These observations suggest cell-mediated immunity is importantin the defense against HPV infection and its associated tumordevelopment. The induction of such immunity might be therapeutic, aswell as prophylactic.

It has been demonstrated that foreign peptides can be incorporated intoviral capsid-like structures and these chimeric particles can be used topresent foreign antigens to the immune system. Published examplesinclude hepatitis B core antigen particle presentation of humanrhinovirus type 2 epitopes (Francis et al. 1990 PNAS USA 87:2545), gp41of HIV (Borisova et al. 1989 FEBS Lett. 259:121), and B19 parvovirusparticle presentation of peptides from herpes simplex virus 1 and murinehepatitis virus (Brown et al. 1994 Virology 198:477). The parvoviruschimeras protected mice from experimental challenge with thecorresponding virus. In all of the above systems, foreign sequences havebeen inserted in proteins integral to the capsid structure and have beenlimited to less than 20 amino acids. However a recent study (Miyamura etal. 1994 PNAS USA 91:8507) has demonstrated that the entire 147 aa henegg white lysozyme protein can be incorporated into B19 parvovirusparticles when fused to the parvovirus L1 minor capsid protein. Thelysozyme remained biologically active and elicited an immune responsewhen injected into rabbits. In perhaps less relevant studies, hepatitisB virus surface antigen particles (which are lipid membrane structures)containing 84 aa of HIV-1 envelope glycoprotein (Michel et al. 1990 J.Virol. 64:2452) and yeast Ty virus-like particles containing a portionof HIV-1 V3 loop (Griffiths et al. 1991 J. Virol. 65:450) have also beenshown to produce an immune response to the inserted peptides wheninoculated into animals. With respect to papillomaviruses, it wasrecently reported that hepatitis B core antigen particles containingHPV16 E7 peptides (all less than 20 aa) induced peptide specificantibodies and T-helper responses in mice (Tindle et al. 1994 Virology200:547).

Chimeric particles based on self-assembled papillomavirus L1 have notbeen reported, nor has the use of L2 as a viral fusion partner forpurposes of generating chimeric VLPs been described. The chimericparticle studies cited above involve viruses that are unrelated topapillomaviruses and thus cannot predict the results of chimericparticle studies involving papillomaviruses. Indeed, in thepapillomavirus study by Kirnbauer et al. 1993 J. Virol. 67:6929, supra,it was demonstrated that a single nonconserved amino acid change in L1is responsible for efficient self-assembly of L1 into VLPs and thepresentation of conformational epitopes, which seem to be required forinduction and detection of clinically relevant immune reactivity. Thus,the studies using viruses unrelated to papillomavirus cannot predictwhether a papillomavirus L2 containing a foreign peptide or protein canco-assemble with papillomavirus L1 into particles, given that a singleamino acid substitution in L1 can abolish efficient self-assembly.Neither can these studies predict whether any resulting chimericparticles will retain the ability to induce or detect neutralizingantibodies or other immune related responses, given that a single aminoacid substitution in L1 can bar the presentation of conformationalepitopes.

It is an object of the present invention to provide chimericpapillomavirus-like particles. These chimeric particles may function asplatforms for multivalent antigen presentation. Or they may serve fordelivery into cells of proteins for processing into peptides andsubsequent presentation of these peptides within the context of MHCmolecules to elicit a cell-mediated immune response. The chimericparticles represent a cost effective way to generate an effectivepapillomavirus vaccine with a broad spectrum of utility. Alternatively,the chimeric papillomavirus-like particles may be applied to VLP and/orfusion partner purification. Or, the particles may operate to deliverinto cells intact and active proteins, for example, enzymes, or toxinsor drugs.

SUMMARY OF THE INVENTION

According to one aspect of the invention, there is provided apapillomavirus-like particle, characterized as having conformationalepitopes, comprising a papillomavirus L1 product and a papillomavirus L2fusion product.

The papillomavirus L2 fusion product may be characterized as being ahuman papillomavirus L2 fusion product or a bovine papillomavirus L2fusion product.

The human papillomavirus L2 fusion product may be characterized as beinga HPV16 L2 fusion product and the bovine papillomavirus L2 fusionproduct may be characterized as being a BPV1 L2 fusion product.

The papillomavirus L2 fusion product may comprise a fusion partnercharacterized as being a peptide or a full-length protein, or maycomprise fusion partners that include peptides or full-length proteinsor combinations thereof linked in tandem.

The fusion partner may be a papillomavirus E6 or E7 product.

The papillomavirus L2 fusion product may be characterized as being fusedat its N-terminus or C-terminus end to a fusion partner.

The papillomavirus L2 fusion product may be characterized as having afusion partner inserted between L2 amino acids.

The papillomavirus L1 product may be characterized as being a humanpapillomavirus L1 product or a bovine papillomavirus L1 product.

The human papillomavirus L1 product may be characterized as being aHPV16 L1 product and the bovine papillomavirus L1 product may becharacterized as being a BPV1 L1 product.

The papillomavirus-like particle may consist essentially of HPV16L1 andHPV16L2-HPV16E7 (full-length), where HPV16E7 is fused to the C-terminusend of HPV16L2, BPVL1 and BPVL2-HPV16E7 (full-length), where HPV16E7 isfused to the C-terminus end of BPVL2, or BPVL1 and BPVL2-HPV16E7 (aminoacids 1-30), where HPV16E7 is fused to BPVL2 between L2 amino acids 274and 275.

According to another aspect of the invention, there is provided one ormore synthetic DNA molecule or molecules characterized as singly ordoubly encoding a papillomavirus L1 product and a papillomavirus L2fusion product where the molecule or molecules direct expression in atransformed host cell of a papillomavirus-like particle, characterizedas having conformational epitopes, comprising the papillomavirus L1product and the papillomavirus L2 fusion product.

The transformed host cell may be an insect host cell (such as Sf9 insecthost cell) and the DNA molecule or molecules may further comprise aninsect cell vector (such as a baculovirus vector), or the transformedhost cell may be a mammalian host cell and the DNA molecule or moleculesmay further comprise a mammalian cell vector (such as a vaccinia virusvector), or the transformed host cell may be a yeast host cell and theDNA molecule or molecules may further comprise a yeast cell vector.

According to another aspect of the invention, there is provided a hostcell transformed with the DNA molecule or molecules of above.

According to yet another aspect of the invention, there is provided amethod for using the DNA molecule or molecules of above comprising thesteps of: providing conditions for the molecule or molecules of above todirect the above expression; and recovering the papillomavirus-likeparticle from the above transformed host cell.

According to still another aspect of the invention, there is provided amethod for producing a papillomavirus-like particle, characterized ashaving conformational epitopes, comprising a papillomavirus L1 productand a papillomavirus L2 fusion product, which method comprises the stepof providing conditions for the DNA molecule or molecules of above todirect the above expression in the above transformed host cell of thepapillomavirus-like particle.

The invention also provides a method of purification of thepapillomavirus-like particle of above comprising the step of exposingthe papillomavirus-like particle to an affinity chromatography column,comprising antibodies that bind to a fusion partner of thepapillomavirus L2 fusion product of the papillomavirus-like particle,resulting in the purification of the particle.

The invention further provides a method of purification of a fusionpartner of the papillomavirus L2 fusion product of thepapillomavirus-like particle of above comprising the step of isolatingthe papillomavirus-like particle of above resulting in the purificationof the fusion partner.

The invention additionally provides a method of delivery into a cell ofa fusion partner of the papillomavirus L2 fusion product of thepapillomavirus-like particle of above comprising the step ofadministering the papillomavirus-like particle of above to the cellresulting in the delivery into the cell of the fusion partner.

The invention moreover provides a vaccine comprising thepapillomavirus-like particle of above.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

This invention arises from the result that a papillomavirus L2 fusionproduct can become incorporated into a papillomavirus L1 product-basedpapillomavirus-like particle that presents conformational epitopes.

This result was unexpected.

In the papillomavirus study by Kirnbauer et al. 1993 J. Virol. 67:6929,supra, it was demonstrated that a single nonconserved amino acid changein L1 is responsible for efficient self-assembly of L1 into VLPs and thepresentation of conformational epitopes, which seem to be required forinduction and detection of clinically relevant immune reactivity. Thus,it could not be predicted whether a papillomavirus L2 containing aforeign peptide or protein could co-assemble with papillomavirus L1 intoparticles, given that a single amino acid substitution in L1 can abolishefficient self-assembly. Neither could it be predicted whether anyresulting chimeric particles would retain the ability to induce ordetect neutralizing antibodies or other immune related responses, giventhat a single amino acid substitution in L1 can bar the presentation ofconformational epitopes.

A papillomavirus L2 fusion product is meant to include a chain of aminoacids in which part of the chain comes from a L2 protein sequence andpart of the chain comes from another protein sequence (or other proteinsequences).

L2 fusion products are produced by splicing (in frame) an open readingframe for one protein (or a number of proteins) next to or into an openreading frame for L2.

Protein engineering is used to determine the structure of the L2 fusionproduct. In a routine exercise for protein engineers, they generatevariants of the natural protein L2. The changes they make can beeducated guesses based on detailed knowledge of the structure of L2;alternatively, changes can be made on a purely random basis. Or acombination of structural information with random mutagenesis andselection can have dramatic results.

For example, the selection of L2 amino acids between which to insert afusion peptide or protein can be made on this basis. The existence of aregion where amino acid sequence and length vary betweenpapillomaviruses suggests that this region represents a structure thatis nonessential for the integrity of L2 and/or its incorporation intoparticles. The observed ability to insert a fusion peptide or proteinwithin L2 implies that such a region exists.

Determination of a structure for the L2 fusion product is performed onthe basis of the ability of the L2 fusion product to become incorporatedinto a papillomavirus L1 product-based papillomavirus-like particle thatpresents conformational epitopes.

Preferred means of determining a structure include assays that measurethe efficiency and/or authenticity of incorporation of an L2 fusionproduct into a papillomavirus L1 product-based papillomavirus-likeparticle.

The efficiency and/or authenticity of formation of L1/L2 VLPs is relatedto the presentation of conformational epitopes (supra).

The chimeric L2 will thus become incorporated into L1-based VLPs with anefficiency and/or authenticity similar to that of incorporation ofwild-type L2.

Other preferred means of determining a structure for the L2 fusionproduct include assays that measure the induction and/or detection ofneutralizing antibodies.

The induction and/or detection of neutralizing antibodies is related tothe presentation of conformational epitopes, because neutralizingantibodies are directed against conformationally dependent epitopes(supra).

The incorporation of the chimeric L2 will thus not inhibit the inductionand/or detection of neutralizing antibodies by chimericpapillomavirus-like particles as compared to L1 or L1/L2 VLPs.

For purposes of the invention, a chimeric L2 fusion product is capableof becoming incorporated into a papillomavirus L1 product-basedpapillomavirus-like particle that presents conformational epitopes,whether identified by the above means or any other means known in theart.

The structure of L2 and its fusion partner (and L1) is meant to includea full-length L2 protein (and L1 protein) and a full-length fusionpartner protein, and their peptide fragments, whether N-terminusfragments, C-terminus fragments, or internal fragments, having at leastabout 20 amino acid residues, advantageously at least about 10 aminoacid residues, and preferably at least about 5 amino acid residues.Type, subgroup and strain variations of L2 (and L1), and human allelicand species variations of the fusion partner protein, are expresslycontemplated as falling within the scope of the invention. The inventionalso includes conservative variants of the full-length L2 protein (andL1 protein) and the full-length fusion partner protein, and theirpeptide fragments, where conservative amino acids are substituted foramino acid residues of wild-type L2 (and L1) and the fusion partnerprotein. To account for degeneracy of the genetic code, the inventionalso includes DNA coding for the same amino acid residues as does theDNA of the L2 (and L1) and fusion partner gene.

The chimeric papillomavirus-like particle itself is envisioned asincorporating any L2 fusion product with any L1 product from anypapillomavirus, whether the genomes are closely related, or aredistantly related, so long as incorporation into particles occurs. Thus,a L2 fusion product, for example, related to any of BPV-1, BPV-2, BPV-4,CRPV, DPV, EEPV, HPV-1, HPV-5, HPV-6, HPV-8, HPV-11, HPV-16, HPV-18,HPV-31 or HPV-33, can be incorporated into particles with any L1product, for example, from any of the above virus, or any type, subgroupor strain variation of papillomavirus.

Because VLPs present conformationally dependent epitopes required forthe induction of high titer neutralizing serum antibodies, VLP chimerascontaining L2 fusion products can operate prophylactically as optimizedsubunit vaccines for the stimulation of humoral immunity to preventpapillomavirus infection and thereby preclude the development ofpapillomavirus associated cancers and other papillomavirus associatedpathologies.

Because it is unlikely VLPs will prove effective as therapeutic vaccinesto induce regression of existing papillomavirus proliferative lesions,as discussed above, chimeric VLPs can function to address this long-feltand heretofore unsatisfied need to develop a therapeutic vaccine.

Chimeric VLPs are expected to bind specific cell surface receptors, getinternalized and be released into the cytoplasm, and thus be more likelyto promote the presentation of peptides in conjunction with class I MHCmolecules for display to cytotoxic T cells for the generation ofcell-mediated immunity. This is in contrast to uncomplexed proteins thatwould not be expected to specifically enter cells, or to promote thepresentation of peptides in the context of Class I MHC molecules toelicit a cytotoxic T cell response (being more likely, if at all, topromote the presentation of peptides to be linked to Class II MHCmolecules and displayed to helper T cells).

Inclusion of one or more fusion partners as L2 fusion products wouldclearly be a cost effective way to generate an effective papillomavirusvaccine with a broad spectrum of utility, including therapy and improvedprevention of clinical lesions.

The fusion partner may be selected from the list consisting of thosefusion partners that would provide a method for expanding the potentialtargets of a VLP-based vaccine, for example, E6 or E7 peptide orfull-length E6 or E7, other papillomavirus peptides or proteins, orpeptides or proteins of other STD or infectious agents, e.g., Herpessimplex, HIV, Chlamydia trachomatis, Neisseria gonorrhoeae, andTreponema pallidum.

The L2 fusion product is not limited to a single fusion partner per L2molecule, and may include additional fusion partners, for example,additional peptides or full-length proteins or combinations thereofderived from the same or different proteins linked in tandem. Also, morethan one L2 fusion product may be co-assembled into a single VLP. Forexample, a L2 fusion product or chimeric VLP containing a viral targetepitope may also be engineered to contain a binding domain of aco-stimulatory protein or an accessory receptor or ligand involved inimmune reactivity, e.g., B7 (which interacts with CD28 on T cells), anintercellular adhesion molecule (ICAM), a lymphocyte functional antigen(LFA), a vascular cell adhesion molecule (VCAM-1), and a heat stableantigen (HSA). (To target the co-stimulators or ligands to the surfaceof VLPs, see Example 14.)

It will be appreciated that the actual preferred amounts of chimericpapillomavirus-like particles as vaccines in the prevention and/ortreatment of disease will vary according to the specific compositionsformulated, the mode of application, the particular situs and theorganism being treated. Dosages for a given host can be determined usingconventional considerations, e.g., by means of an appropriate,conventional vaccination protocol.

Alternatively, chimeric VLPs can used in a method of purification ofVLPs. VLPs are useful per se as prophylactic vaccines and inimmunodiagnostics (supra). Briefly, antibodies to the fusion partner aregenerated using standard immunological techniques, an affinitychromatography column is constructed using the antibodies, and the VLPsare subsequently purified in an affinity chromatography step.

Or, chimeric VLPs can be used in a method of purification of a fusionpartner. For example, VLPs containing a L2-E7 fusion product may beuseful as a means of obtaining correctly folded E7. It has been reportedthat a greater percentage of cervical cancer patients are seropositivefor conformationally correct E7 than for denatured E7, as isolated frombacteria (Viscidi et al. 1993 Int. J. Cancer 55:780). The in vitrotranscription-translation system used to generate E7 in this report islaborious and expensive, and uses radio-labeled E7. It would beadvantageous to purify chimeric E7 VLPs and use the material in an E7ELISA. To avoid complications of seroreactivity with human L1 or L2,BPV-based particles could be used. Monitoring E7 seroreactivity, whichcorrelates with cancer as opposed to premalignant disease, has beenproposed to be useful to follow the course of disease in cervical cancerpatients and to screen for reoccurrences (id.).

Optionally, chimeric VLPs can be used in a method of delivery of anintact and active protein into cells. This protein may be, for example,an enzyme, or a toxin or a drug. The chimeric VLPs are administered tothe cells, either in vitro, in vivo, in situ, or ex vivo, and theprotein is subsequently delivered into the cell where it functions forits intended purpose, for example, as an enzyme, or a toxin or a drug.

We have succeeded at generating chimeric papillomavirus-like particlesincorporating E7 protein or peptide fused to L2.

A papillomavirus E7 gene when fused in frame to the 3′ end of the L2gene or within part of the L2 open reading frame expressed an L2-E7fusion protein that, in the presence of L1 protein, was incorporatedinto virus-like particles. Efficiency and authenticity of incorporationof L2-E7 into VLPs was similar to that of the wild-type L2 protein. BPVL1/L2-E7 virus-like particles were found to induce neutralizingantibodies as effectively as BPV L1/L2 particles. Chimeric particleswere observed to elicit humoral immunity specific for fusion partnerepitopes in that rabbits immunized with L2-E7 chimeric particlesgenerated antibodies directed against E7.

Having succeeded at generating chimeric papillomavirus-like particlesopens the door to fusing virtually any protein or peptide to an L2product for incorporation into chimeric papillomavirus-like particles.

We accomplished the production of chimeric VLPs by generating L2-E7fusion proteins and expressing these proteins along with L1 viarecombinant baculoviruses in insect cells. We generated three chimericVLPs: HPV16L1+HPV16L2-HPV16E7 (full-length), BPVL1+BPVL2-HPV16E7(full-length) and BPVL1+BPVL2-HPV16E7 (aa 1-30).

BPV L2 was fused at its C-terminus end to HPV16 E7. HPV16 L2 was fusedat its C-terminus end to HPV16 E7. Additionally, the first 30 codons ofHPV16 E7 were inserted between codons 274 and 275 of BPV L2.

Sucrose gradient co-sedimentation and co-immunoprecipitation of L1 andthe L2-E7 chimeras demonstrated the incorporation of the chimericprotein into the VLPs.

Transmission electron microscopy revealed that particles containing theL2-E7 fusion protein assembled with the same efficiency as L1 or L1/L2VLPs, and were morphologically indistinguishable.

In vitro BPV1 neutralization assays demonstrated that the BPVL1-containing chimeric VLPs were capable of inducing neutralizingantisera. Titers were comparable to those obtained using BPV L1/L2 VLPs.Equivalent neutralizing titers of 30,000 were obtained for both BPVL1/L2VLPs and BPVL1/L2-HPV16E7 (full-length) chimeric VLPs.

Rabbits immunized with L2-E7 chimeric particles generated antibodiesdirected against E7, demonstrating the induction by chimeric VLPs ofhumoral immunity specific for fusion partner epitopes, and alsoestablishing the location of E7 epitopes as external to VLPs.

As set forth in Example 1, chimeric recombinant baculoviruses can begenerated. Genes for the chimeras may be obtained from genomic sourcesor cDNAs, by direct synthesis, or by any combination thereof. L2 and itsfusion partner genes can be amplified by recombinant PCR techniques, forexample, with oligos containing restriction enzyme sites andcomplementarities that facilitate fusion and cloning into expression,transfer and/or cloning vectors, e.g., plasmids.

Fused genes can be cloned into a baculovirus expression vector. Anotherbaculovirus expression vector containing L1 can be generated. Or thefused genes can be cloned into a baculovirus double expression vectorthat already contains L1 .

Example 2 sets forth a typical procedure for selection of recombinantbaculoviruses. CsCl-purified (or equivalent) recombinant plasmid can becotransfected with baculovirus DNA into Sf9 insect cells by usingLipofectin (or equivalent). The recombinant baculoviruses can then beplaque-purified (for example) using conventional baculovirus vector andinsect cell culture procedures.

It may be advantageous to use two single expression baculovirus vectorsinstead of a double expression baculovirus vector. In this case,chimeric VLPs can be produced by infecting Sf9 cells with tworecombinant baculoviruses, one encoding a L1 product and the otherencoding the L2 fusion product. Locating the genes on different vectorsfacilitates manipulation of the amount of L1 product and L2 fusionproduct produced. This approach permits one to change the ratio of L2fusion product to L1 product in a VLP. Although in native virions, L2 isthe minor component as compared to L1, we can achieve greaterincorporation of L2 fusion product into VLPs using two single expressionvectors.

As set forth in Example 3, chimeric particles can be purified by bandingin cesium chloride (or equivalent). Sf9 insect cells can be infected,for example, at a multiplicity of infection of 10 with recombinantbaculoviruses. After 72 hours (or so), cells can be harvested andsonicated in a phosphate-buffered saline (or equivalent) for 60 sec (orso). After low speed clarification (or equivalent), the lysates can besubjected to centrifugation, for example, at 110,000×g for 2.5 h througha 40% (wt/vol) sucrose/PBS cushion (SW-28 rotor). The resuspendedpellets can be centrifuged to equilibrium, for example, at 141,000×g for20 h at room temperature in a 10-40% (wt/wt) CsCl/PBS gradient. Thevisible band can be harvested, centrifuged to equilibrium again by usingthe identical conditions, dialyzed extensively against PBS, and storedat 4° C. (for example).

As set forth in Example 4, co-sedimentation of chimeric complexes can beestablished, for example, by analytical gradient centrifugation. E.g., a12 to 45% sucrose step gradient can be allowed to linearize overnight at4° C., dialyzed samples can be layered on top, and the gradient can becentrifuged at 41,000 rpm (288,000×g) for 2 h in a SW-41 rotor.Fractions can be harvested and analyzed for co-sedimentation, forexample, by Western blotting or co-immunoprecipitation.

As set forth in Example 5, co-sedimentation can be established, forexample, by co-immunoprecipitation.

As set forth in Example 6, antisera can be produced. This can be done toconduct a BPV1 neutralization assay (or equivalent) as is described inDvoretsky et al., Virology 103:369 (1980). E.g., antisera can beproduced as follows. Rabbits can be immunized by subcutaneous injectionof 330 μl of CsCl gradient-purified particles in PBS at a concentrationof 1 mg/ml. Rabbits can then be boosted with the same amount ofparticles two weeks and four weeks after the primary injection.

As set forth in Example 7, a BPV1 neutralization assay (or equivalent)can be performed to test whether BPV chimeric particles presentconformational epitopes. E.g., serial dilutions of sera obtained 3 weeksafter the second booster injection can be incubated with ≈500focus-forming units of BPV1 virus for 30 min, the virus can be adsorbedto C127 cells for 1 h, and the cells can be cultured for 3 weeks. Focican then be stained with 0.5% methylene blue/0.25% carbol fuchsin inmethanol. Neutralizing titers can be obtained for chimeric VLPs andcontrol BPVL1-L2 VLPs. Equivalent neutralizing titers will indicateproper folding of the chimeric particles effective to presentconformational epitopes.

As set forth in Example 8, chimeric particles can be assayed, forexample, by transmission electron microscopy. E.g., purified particlescan be adsorbed to carbon-coated grids, stained with 1% uranyl acetate,and examined with a Philips electron microscope model EM 400T at ×36,000magnification. Efficiency of incorporation and morphology of chimericparticles and L1 or L1/L2 VLPs can be compared and contrasted.Indistinguishable efficiency of incorporation and morphology willindicate proper self-assembly of the chimeric particles.

As set forth in Example 9, chimeric particles can be assayed, forexample, for induction of humoral immunity specific for fusion partnerepitopes. E.g., rabbits can be inoculated with chimeric VLPs. The seracan be analyzed for antibodies, for example, by subjecting a sample ofthe fusion partner antigen to SDS-PAGE and then Western blotting. Immuneand preimmune (control) sera can be applied at an appropriate dilution.Detection of the fusion partner band in the Western blot by serum fromthe immune rabbit indicates the induction of antibodies specific forfusion partner epitopes.

As set forth in Example 10, chimeric particles can be assayed, forexample, for induction of cell-mediated immunity specific for fusionpeptides, for instance, by injecting chimeric VLPs into mice, andmeasuring antigen-specific T cell proliferation.

As set forth in Example 11, chimeric particles can be assayed, forexample, for induction of prophylactic immunity against challengeinfection, for instance, by immunizing rabbits with chimeric CRPV VLPsand nonchimeric CRPV VLPs (control), and subsequently challenging withinfectious CRPV, to demonstrate that L2 fusions do not abrogateprophylactic immunity; or by immunizing experimental animals withchimeric VLPs containing an STD agent, subsequently challenging with theSTD agent, and measuring increased survival against lethal challenge ordecreased infection following sub-lethal challenge.

As set forth in Example 12, chimeric particles can be assayed, forexample, for induction of therapeutic immunity against pre-existingpapillomas, for instance, by immunizing rabbits (that have pre-existingpapillomas) using chimeric CRPV VLPs and nonchimeric CRPV VLPs(control), and measuring regression of the pre-existing papillomas.

As set forth in Example 13, chimeric particles can be assayed, forexample, for immunotherapy and immunoprevention of tumors.

Particular aspects of the invention may be more readily understood byreference to the following examples, which are intended to exemplify theinvention, without limiting its scope to the particular exemplifiedembodiments.

Example 1 Generation of Chimeric Recombinant Baculoviruses

Three chimeras were generated: HPV16L2-HPV16E7, BPVL2-HPV16E7, andBPVL2-HPV16E7 (aa 1-30). HPV16L2-HPV16E7 contained the full-lengthHPV16E7 fused to the 3′ (aa 473) of HPV16L2. BPVL2-HPV16E7 contained thefull-length HPV16E7 fused to the 3′ of BPVL2 (aa 469). BPVL2-HPV16E7 (aa1-30) contained the first 30 amino acids of HPV16E7 fused to the middleof BPVL2 between amino acids 274 and 275.

L2-E7 chimeric genes were generated via recombinant PCR techniques(Higuchi, R. (1990) in PCR Protocols; A Guide to Methods andApplications, ed. Innis, Mass., Gelfand, D H, Sninsky, J J, and White, TJ., Academic, New York, pp. 177-180). For the chimeras containingfull-length HPV16E7, L2 was amplified with a 5′ oligo containing arestriction enzyme site, and a 3′ oligo which was complementary to theE7 5′ oligo. In an independent reaction, E7 was amplified with a 5′oligo complementary to the L2 3′ oligo and a 3′ oligo containing arestriction enzyme site. The L2 and E7 genes were then fused in a secondprimer extension reaction using only the outside (L2 5′, and E7 3′)oligos. For the chimera containing only amino acids 1-30 of HPV16E7, the“internal” primers encoded the first 30 aa of HPV16E7.

The fused, genes were then cloned into the baculovirus double expressionvector pSynwtVI-, (which already contained L1 cloned under thepolyhedrin promoter (Kirnbauer et al. 1993 J. Virol. 67:6929)immediately downstream of the pSyn promoter.

The BPVL2-HPV16E7 chimeras were cloned as 5′ BglII to 3′BglII fragments.The HPV16L2-HPV16E7 chimera was cloned as a 5′ SstII to 3′ SstIIfragment. The primers used for BPVL2-HPV16E7 (full-length) were:

BPVL2 sense,

5′GCGGTAGATCTACCTATAAATATGAGTGCACGAAAAAGAGTAAAACGT3′, (SEQ ID NO: 1)and

antisense,

(SEQ ID NO: 2) 5′GCAATGTAGGTGTATCTCCATGCATGGCATGTTTCCGTTTTTTTCGTTTCCTCAACAAGGAGGG3′,

HPV16E7, sense,

(SEQ ID NO: 3) 5′CCCTCCTTGTTGAGGAAACGAAAAAAACGGAAACATGCCATGCATGGAGATACACCTACATTGC3′,and

antisense,

(SEQ ID NO: 4) 5′CCGCTAGATCTGGTACCTGCAGGATCAGCCATGG3′.

The primers used for BPVL2-HPV16E7 (aa 1-30) were as follows: BPVL2,sense: same as above.

Internal primer, antisense,

(SEQ ID NO: 5) 5′CAGTTGTCTCTGGTTGCAAATCTAACATATATTCATGCAATGTAGGTGTATCTCCATGCATGGATGAAAACACTTCAGGATCTTCCGTGGGC3′.

Internal primer, sense,

(SEQ ID NO: 6) 5′GCATGAATATATGTTAGATTTGCAACCAGAGACAACTGATCTCTACTGTTATGAGCAATTAAATGACCAAACATTTGCAAACCCACTGTATGAAGCAG AACC3′.

BPVL2, antisense,

5′CCGCTAGATCTAGGGAGATACAGCTTCT GGCCTTGTTGCCACAACGC3′. (SEQ ID NO: 7)

The primers used for HPV16L2-HPV16E7 chimeras were as follows:

HPV16L2, sense,

5′GCGGTCCGCGGAATATGCGACACAAACGTTCTGCAAAACGCACAAAACGT3′, (SEQ ID NO: 8)

and antisense,

5′ATCTCCATGCATGGCAGCCAAAGAGAC3′, (SEQ ID NO: 9)

HPV16E7, sense,

5′GTCTCTTTGGCTGCCATGCATGGAGAT3′, (SEQ ID NO: 10)

and antisense,

5′GCTCCGCGGGGTACCTGCAGGATCAGCC3′. (SEQ ID NO: 11)

Example 2 Selection of Recombinant Baculoviruses

CsCl-purified recombinant plasmid was cotransfected with baculovirus DNA(Baculo-Gold; PharMingen, San Diego, Calif.) into Sf9 insect cells(ATCC, CRL 1711) by using Lipofectin (GIBCO/BRL, Gaithersburg, Md.)(Hartig, P. C. Biotechniques 11:310 (1991)). The recombinantbaculoviruses were plaque-purified as described (Summers, M. D. & Smith,G. E. (1987) A Manual of Methods for Baculovirus Vectors and Insect CellCulture Procedures, Texas Agricultural Experiment Station Bulletin (Tex.Agric. Exp. Stn., College Station, Tex.), Vol 1555).

Example 3 Purification of Chimeric Particles

Sf9 insect cells were infected at a multiplicity of infection of 10 withrecombinant baculoviruses. After 72 h, the harvested cells weresonicated in phosphate-buffered saline (PBS) for 60 sec. After low-speedclarification, the lysates were subjected to centrifugation at 110,000×gfor 2.5 h through a 40% (wt/vol) sucrose/PBS cushion (SW-28 rotor). Theresuspended pellets were centrifuged to equilibrium at 141,000×g for 20h at room temperature in a 10-40% (wt/wt) CsCl/PBS gradient. The visibleband was harvested, centrifuged to equilibrium again by using theidentical conditions, dialyzed extensively against PBS, and stored at 4°C.

Example 4 Co-Sedimentation of L1/L2-E7 Complexes

To determine whether the L2-E7 fusion protein was incorporated intoparticles, analytical gradient centrifugation was performed aspreviously described for determining the co-assembly of L1 and L2(Kirnbauer et al. 1993 J. Virol. 67:6929).

Briefly, a 12 to 45% sucrose step gradient was allowed to linearizeovernight at 4° C., dialyzed samples were layered on top, and thegradient was centrifuged at 41,000 rpm (288,000×g) for 2 h in a SW-41rotor. Fractions were harvested. The fractions were analyzed byCoomassie stained SDS-PAGE (L1) or Western blotting with an anti-BPVL2polyclonal Ab, or anti-HPV16E7 polyclonal Ab, and ¹²⁵I-labeledanti-rabbit IgG.

Association of the chimeric L2-E7 with the virus-like particles wasestablished by co-sedimentation.

Example 5 Co-Immunoprecipitation of L1/L2-E7 Complexes

To obtain evidence that the L2-E7 fusion proteins formed stablecomplexes with L1, co-immunoprecipitation experiments were performed.

Briefly, BPVL1/L2-HPV16E7 (full-length) and BPVL1/L2-HPV16E7 (aa 1-30)VLP preparations were immunoprecipitated in PBS, 1% Triton® X-100 withanti-L1 Mab 5B6 (Roden et al., J. Virol., in press, (November, 1994)),an anti-L1 polyclonal Ab, pre-immune serum or an irrelevant Ab (anti-E1AMab) and protein A-Sepharose and subjected to SDS-PAGE. Proteins wereimmunoblotted and probed with anti-BPVL2, or anti-HPV16E7 sera, or ananti-HPV16E7 Mab (Triton Diagnostics, Alameda, Calif.).

Association of the chimeric L2-E7 with the virus-like particles wasestablished by co-immunoprecipitation.

Example 6 Production of Antisera

Rabbits were immunized by subcutaneous injection of 330 μl of CsClgradient-purified particles in PBS at a concentration of 1 mg/ml.Rabbits were boosted with the same amount of particles two weeks andfour weeks after the primary injection.

Example 7 BPV1 Neutralization Assay

A focus-forming assay was performed as described (Kirnbauer et al. 1992PNAS USA 89:12180).

Briefly, serial dilutions of rabbit sera obtained 3 weeks after thesecond booster injection were incubated with ≈500 focus-forming units ofBPV1 virus for 30 min, the virus was adsorbed to C127 cells for 1 h, andthe cells were cultured for 3 weeks (Dvoretzky et al. 1980 Virology103:369). The foci were stained with 0.5% methylene blue/0.25% carbolfuchsin in methanol.

Equivalent neutralizing titers of 30,000 were obtained for the BPVL1/L2VLPs and BPVL1/L2-HPV16E7 (full-length) chimeric VLPs.

Example 8 Electron Microscopy

Transmission electron microscopy was performed as described (Kirnbaueret al. 1992 PNAS USA 89:12180)

Briefly, purified particles were adsorbed to carbon-coated grids,stained with 1% uranyl acetate, and examined with a Philips electronmicroscope model EM 400T at ×36,000 magnification.

Particles containing the L2-E7 fusion protein were found to beindistinguishable from L1 or L1/L2 VLPs in terms of morphology andefficiency of incorporation.

Example 9 Induction of Antibodies

Rabbits inoculated with BPVL1/L2-HPV16E7 papillomavirus-like particlesproduced antisera that recognized E7.

To analyze the sera for E7 specificity, 2.5 μg of HPV16E7 and 2.5 μg ofBSA (control) were subjected to SDS-PAGE and then Western blotted.Immune and preimmune (control) sera were applied at a dilution of 1:10.

Serum from the immune rabbit specifically detected the HPV16E7 proteinband in the Western blot, indicating the induction of antibodiesspecific for E7 epitopes.

Example 10 Induction of Cell-Mediated Immunity

Chimeric particles are assayed for induction of cell-mediated immunityspecific for E7 peptides, for example, by injecting chimeric VLPs intomice, and measuring antigen-specific T cell proliferation.

Example 11 Prophylactic Immunity

Chimeric particles are assayed for induction of prophylactic immunityagainst challenge infection, for example, by immunizing rabbits with E7chimeric CRPV VLPs and nonchimeric CRPV VLPs (control), and subsequentlychallenging with infectious CRPV, to demonstrate that L2-E7 fusions donot abrogate prophylactic immunity; or by immunizing experimentalanimals with chimeric VLPs containing an STD agent, subsequentlychallenging with the STD agent, and measuring increased survival againstlethal challenge or decreased infection following sub-lethal challenge.

Example 12 Therapeutic Immunity

Chimeric particles are assayed for induction of therapeutic immunityagainst pre-existing papillomas, for example, by immunizing rabbits(that have pre-existing papillomas) using E7 chimeric CRPV VLPs andnonchimeric CRPV VLPs (control), and measuring regression of thepre-existing papillomas.

Example 13 Immunotherapy and Immunoprevention of Tumors

Chimeric particles are assayed for the ability to prevent tumordevelopment or to treat existing tumors, for example, in experimentalanimals such as mice using tumor cells expressing, e.g., E7. Animals areimmunized with, e.g., L2-E7 chimeric VLPs and tested for growth ofinoculated tumorigenic cells that express, e.g., E7. This approach hasbeen shown to work for animals immunized with noncomplexed E7 and aco-stimulatory protein (Chen et al. 1990 PNAS USA 88:110).

Example 14 Targeting the Surface of Chimeric VLPs

It may be desirable for certain molecules, such as co-stimulators orligands that bind to cell-surfaces, to be present on the surface ofchimeric VLPs. Roden et al., J. Virol., in press, (November, 1994)determined that regions of L2 are located on the surface of VLPs. Apeptide composed of BPV L2 amino acids 44-173, when inoculated intorabbits, induced neutralizing antibodies that were active at a 1:1000serum dilution. These results indicate that regions of L2 specifyneutralizing epitopes, and that these regions are therefore accessibleto the surface of native virions. Consequently, fusing a non-L2polypeptide to one of these regions would likely result in the targetingof this polypeptide to the surface of a chimeric VLP.

While particular embodiments of the invention have been described indetail, it will be apparent to those skilled in the art that theseembodiments are exemplary rather than limiting, and the true scope ofthe invention is that defined within the appended claims.

1. A papillomavirus-like particle, characterized as havingconformational epitopes, comprising a papillomavirus L1 product and apapillomavirus L2 fusion product. 2.-10. (canceled)